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Dendritic cell (DC) homeostasis, like all mature blood cells, is maintained via hierarchal generation from hematopoietic precursors; however, little is known about the regulatory mechanisms governing DC generation. Here, we show that prostaglandin E(2) (PGE(2)) is required for optimal Flt3 ligand-mediated DC development and regulates expression of the Flt3 receptor on DC-committed progenitor cells. Inhibition of PGE(2) biosynthesis reduces Flt3-mediated activation of STAT3 and expression of the antiapoptotic protein survivin, resulting in increased apoptosis of DC-committed progenitor cells. Reduced DC development caused by diminished PGE(2) signaling is reversed by overexpression of Flt3 or survivin in DC progenitors and conversely is mimicked by STAT3 inhibition. PGE(2) regulation of DC generation is specifically mediated through the EP1 and EP3 G protein PGE(2) receptors. These studies define a novel DC progenitor regulatory pathway in which PGE(2) signaling through EP1/EP3 receptors regulates Flt3 expression and downstream STAT3 activation and survivin expression, required for optimal DC progenitor survival and DC development in vivo.

A complex therapeutic challenge for Alzheimer's disease (AD) is minimizing deleterious aspects of microglial activation while maximizing beneficial actions, including phagocytosis/clearance of amyloid beta (Abeta) peptides. One potential target is selective suppression of microglial prostaglandin E(2) receptor subtype 2 (EP2) function, which influences microglial phagocytosis and elaboration of neurotoxic cytokines. To test this hypothesis, we transplanted bone marrow cells derived from wild-type mice or mice homozygous deficient for EP2 (EP2(-/-)) into lethally irradiated 5-month-old wild-type or APPswe-PS1DeltaE9 double transgenic AD mouse model recipients. We found that cerebral engraftment by bone marrow transplant (BMT)-derived wild-type or EP2(-/-) microglia was more efficient in APPswe-PS1DeltaE9 than in wild-type mice, and APPswe-PS1DeltaE9 mice that received EP2(-/-) BMT had increased cortical microglia compared with APPswe-PS1DeltaE9 mice that received wild-type BMT. We found that myeloablative irradiation followed by bone marrow transplant-derived microglia engraftment, rather than cranial irradiation or BMT alone, was responsible for the approximate one-third reduction in both Abeta plaques and potentially more neurotoxic soluble Abeta species. An additional 25% reduction in cerebral cortical Abeta burden was achieved in mice that received EP2(-/-) BMT compared with mice that received wild-type BMT. Our results provide a foundation for an adult stem cell-based therapy to suppress soluble Abeta peptide and plaque accumulation in the cerebrum of patients with AD.

Prostaglandin E2 is one of several eicosanoid products of the cyclooxygenase isozymes and is a key regulator of innate immune responses; it also possesses paracrine effects on mature neurons. The prostaglandin E2 receptor family consists of four subtypes of which EP1 and EP2 are known to be expressed by microglia. Lipopolysaccharide (LPS)-induced innate immune activation leads to the degeneration of intermediate progenitor cells (IPCs) that are destined for neuronal maturation in the hippocampal subgranular zone (SGZ); these cells can be identified by the expression of the transcription factor T-box brain gene 2 (Tbr2). Importantly, depletion of LPS-induced IPCs from the SGZ is suppressed by cyclooxygenase inhibitors. We therefore tested the hypothesis that either EP1 or EP2 is critical to LPS-induced depletion of Tbr2+ IPCs from the SGZ. Expression of either EP1 or EP2 was necessary for Toll-like receptor 4-dependent innate immune-mediated depletion of these Tbr2+ IPCs in mice. Moreover, EP1 activation was directly toxic to murine adult hippocampal progenitor cells; EP2 was not expressed by these cells. Finally, EP1 modulated the response of murine primary microglia cultures to LPS but in a manner distinct from EP2. These results indicate that prostaglandin E2 signaling via either EP1 or EP2 is largely to completely necessary for Toll-like receptor 4-dependent depletion of IPCs from the SGZ and suggest further pharmacological strategies to protect this important neurogenic niche.

Ultraviolet (UV) light is a complete carcinogen inducing and promoting squamous-cell carcinoma (SCC) of the skin. Recent work has shown that SCC initiation and promotion are enhanced by prostaglandin E2 (PGE2). PGE2 interacts with specific EP receptors to regulate cellular functions. Previous work from our group has shown that the prostaglandin E2 EP2 receptor is a powerful regulator of keratinocyte growth. SKH-1 hairless mice lacking the EP2 receptor were therefore studied to understand how this growth signaling pathway contributes to photocarcinogenesis. Our data indicate that UV-irradiated mice lacking EP2 receptors exhibit decreased proliferation and a poor capacity for epidermal hypertrophy in response to UV injury. In a chronic irradiation model, these animals were protected from tumor formation, developing 50% fewer tumors than wild-type controls. Despite this capacity to protect against tumorigenesis, animals lacking EP2 receptors grew tumors that were larger in size, with a more aggressive phenotype. Further study suggested that this susceptibility may be associated with synthesis of active metalloproteinase enzymes in greater quantities than keratinocytes expressing the EP2 receptor, thereby enhancing the invasive potential of EP2-/- cells.

Experimental therapies for Alzheimer's disease (AD) are focused on enhanced clearance of neurotoxic Abeta peptides from brain. Microglia can be neuroprotective by phagocytosing Abeta; however, this comes at the cost of activated innate immunity that causes paracrine damage to neurons. Here, we show that ablation of E prostanoid receptor subtype 2 (EP2) significantly increased microglial-mediated clearance of Abeta peptides from AD brain sections and enhanced microglial Abeta phagocytosis in cell culture. The enhanced phagocytosis was PKC-dependent and was associated with elevated microglial secretion of the chemoattractant chemokines, macrophage inflammatory protein-1alpha and macrophage chemoattractant protein-1. This suggested that microglial activation is negatively regulated by EP2 signaling through suppression of prophagocytic cytokine secretion. However, despite this enhancement of Abeta phagocytosis, lack of EP2 completely suppressed Abeta-activated microglia-mediated paracrine neurotoxicity. These data demonstrate that blockade of microglial EP2 is a highly desirable mechanism for AD therapy that can maximize neuroprotective actions while minimizing bystander damage to neurons.

Background: Prostaglandin E2 (PGE2) plays an important role in many physiologic and pathophysiologic processes in the kidney. Multiple enzymes are involved in PGE2 biosynthesis, including phospholipases, cyclooxygenases (COX), and the PGE2 synthases (PGES). The present studies were aimed at determining the intrarenal localization of mPGES-1 and whether it is coexpressed with COX-1 or COX-2., Methods: Rabbit mPGES-1 and COX-1 cDNAs were cloned using reverse transcription-polymerase chain reaction (RT-PCR) and screening a cDNA library. RNase protection assay and immunoblotting were used to examine mPGES-1 expression levels. In situ hybridization and immunostaining were used to determine the intrarenal localization of mPGES-1 and cyclooxygenases., Results: Rabbit mPGES-1 shares high sequence similarity to the human homolog. Nuclease protection studies showed that the kidney expresses among the highest level of mPGES-1 of any rabbit tissue. In situ hybridization showed COX-1 and mPGES-1 mRNA was highly expressed in renal medullary collecting ducts (MCD), and to a lesser extent in cortical collecting ducts (CCD). Fainter mPGES-1 expression was also observed in macula densa (MD) and medullary interstitial cells (RMICs), where COX-2 is highly expressed. Double-labeling studies (immunostaining plus in situ hybridization) and immunohistochemistry of mouse tissues confirmed that mPGES-1 predominantly colocalizes with COX-1 in distal convoluted tubule (DCT), CCD, and MCD, and is coexpressed with COX-2 at lower levels in MD and RMICs., Conclusion: Together, these studies suggest mPGES-1 colocalizes with both COX-1 and COX-2 to mediate the biosynthesis of PGE2 in the kidney.

The present study examined the expression and role of the thiazolidinedione (TZD)-activated transcription factor, peroxisome proliferator-activated receptor gamma (PPARgamma), in human bladder cancers. In situ hybridization shows that PPARgamma mRNA is highly expressed in all human transitional epithelial cell cancers (TCCa's) studied (n=11). PPARgamma was also expressed in five TCCa cell lines as determined by RNase protection assays and immunoblot. Retinoid X receptor alpha (RXRalpha), a 9-cis-retinoic acid stimulated (9-cis-RA) heterodimeric partner of PPARgamma, was also co-expressed in all TCCa tissues and cell lines. Treatment of the T24 bladder cancer cells with the TZD PPARgamma agonist troglitazone, dramatically inhibited 3H-thymidine incorporation and induced cell death. Addition of the RXRalpha ligands, 9-cis-RA or LG100268, sensitized T24 bladder cancer cells to the lethal effect of troglitazone and two other PPAR- activators, ciglitazone and 15-deoxy-delta(12,14)-PGJ2 (15dPGJ(2)). Troglitazone treatment increased expression of two cyclin-dependent kinase inhibitors, p21(WAF1/CIP1) and p16(INK4), and reduced cyclin D1 expression, consistent with G1 arrest. Troglitazone also induced an endogenous PPARgamma target gene in T24 cells, adipocyte-type fatty acid binding protein (A-FABP), the expression of which correlates with bladder cancer differentiation. In situ hybridization shows that A-FABP expression is localized to normal uroepithelial cells as well as some TCCa's. Taken together, these results demonstrate that PPARgamma is expressed in human TCCa where it may play a role in regulating TCCa differentiation and survival, thereby providing a potential target for therapy of uroepithelial cancers.

To elucidate roles of protein kinase C (PKC) zeta in rabbit kidney, PKC zeta was cloned from a rabbit kidney cortex cDNA library. Sequencing revealed a 2113 m insert with an open reading frame encoding a protein of 591 amino acids. The predicted amino acid sequence is 93.7% identical with rat PKC zeta. In situ hybridization in rabbit kidney with a riboprobe generated from the cloned cDNA, showed PKC zeta mRNA is highly expressed in proximal tubule, thick limb, and collecting duct. No message was detected over glomerular cells. Immunohistochemical studies using a monoclonal antibody against PKC zeta confirmed this distribution with low expression in vascular elements and high expression in tubule epithelium. Confocal microscopy showed diffuse cytosolic immunoreactivity in confluent cultured cortical collecting ducts (CCDs). However, in subconfluent cells, immunoreactivity was restricted to the peri-nuclear area. This differential distribution of PKC zeta in the CCD suggests that PKC zeta action be involved in growth and differentiation of the collecting duct. In conclusion, PKC zeta is differentially expressed in the rabbit kidney with high expression in the tubule epithelium and little expression in vascular elements. These studies suggest an important role for PKC zeta along the nephron.

The physiological effects of PGE2 appear to be mediated by at least three different "E-prostanoid" receptors designated EP1,EP2, and EP3. These receptors are differentially activated by structural PGE analogs (such as misoprostol) and each couples to a different signal transduction mechanism. Studies demonstrating that inhibition of water absorption in the collecting duct is mediated by a Gi coupled mechanism, suggests that an EP3 receptor is involved the renal effects of PGE2. We used in situ hybridization to determine the tissue distribution of the rabbit EP3 receptor. [alpha-35S] UTP labeled antisense RNA, comprising transmembrane domains IV through VII, was hybridized to tissue sections. Specific labeling of kidney, stomach and adrenal was observed. In the kidney, medullary thick ascending limb and cortical and medullary collecting ducts were intensely labeled, while no labeling of glomeruli, proximal tubules, or cortical thick ascending limbs was observed. The adrenal gland labeled exclusively in the medulla. In the stomach the gastric epithelial crypts were the predominant site of hybridization, without evidence of labeling of the smooth muscle. These results suggest an important role for the EP3 receptor in mediating PGE2 effects in these tissues.